We are continuing our study of the E. Coli bacteriophage T4 model system for duplex DNA replication in which efficient DNA replication in vitro is achieved with purified proteins encoded by T4 phage: T4 DNA polymerase (gene 43), gene 32 DNA helix-destabilizing protein, the gene 44/62 and gene 45 polymerase accessory proteins, the genes 41, 61, and 59 primase-helicase, RNase H, and DNA ligase. We are collaborating with Tim Meuser and Craig Hyde, NIAMS, to determine the structure of the T4 DNA replication proteins by x-ray diffraction. T4 RNase is a 5' to 3' exonuclease that removes RNA primers from the lagging strand of the DNA replication fork, and is a member of the RAD2 family of eukaryotic and prokaryotic replication and repair nucleases. The crystal structure of the full-length native form of T4 RNase H has been solved at 2.06 angstroms maximal resolution in the presence of Mg2+, but in the absence of nucleic acids. The most conserved residues are clustered together in a large cleft with two Mg2+ in the proposed active site of the enzyme. The T4 RNase H structure suggests how the widely separated conserved regions in the larger nucleotide excision repair proteins such as human XPG could assemble into a structure like that of the smaller replication nucleases. Site-directed mutagenesis of the presumptive active site residues of T4 RNase H shows that the D19N mutant protein has almost no nuclease activity, but continues to bind double-stranded DNA. This mutant protein is being used for cocrystallization of T4 RNase H with it substrate. The D200N mutant retains the nuclease, whereas D157N has greatly reduced activity. T4 RNase H is strongly stimulated and becomes processive in the presence of the T4 gene 32 DNA binding protein. A truncated RNase H, which is missing the C-terminal 27 amino acids that form two ` helices, makes the first cut from the 5' end, but has reduced ability to continue degradation. This truncated enzyme is not stimulated by 32 protein, suggesting a possible role for this C-terminal region. We are studying the mechanism by which the gene 59 protein stimulates DNA unwinding by the 41 helicase, and primer synthesis dependent on both the 41 and 61 proteins, using affinity chromatography, chemical cross-linking, and mutagenesis. The solution of the crystal structure of the gene 59 helicase assembly protein is in progress.